This program explores innate immune, pro-inflammatory, and other signaling pathways linked to deliberate reactive oxygen species (ROS) production by Nox family NADPH oxidases. The prototypical NADPH oxidase complex of circulating phagocytes (Nox2-based) is well known for its functions in microbial killing. The non-phagocytic oxidases (Nox1, Nox4, Duox1, Duox2) are expressed primarily in epithelial cells, notably on mucosal surfaces (lung and gastrointestinal tract), in liver, kidney, thyroid and exocrine glands (salivary, mammary), and in vascular tissues. ROS produced by these oxidases affect cell migration, proliferation, wound healing, tumor invasiveness and metastasis, cell differentiation, senescence, programmed cell death (apoptosis), oxygen sensing, and responses to growth factors, cytokines, hormones or danger- and pathogen-associated molecular patterns (DAMPs and PAMPs). Despite differences in their induction, regulation, and responsiveness to diverse signals, these enzymes share a common general function of maintenance and defense of epithelial barriers. In 2015, we explored the function of epithelial cell NADPH oxidases in three pathological or physiological settings: 1) Nox4 induction by transforming growth factor-beta (TGF-beta) and its potential roles in profibrotic and prometastatic disease, 2) Nox1 regulation and function in colon epithelial cell migration, and 3) Duox function in respiratory epithelial cells infected with influenza A virus (IAV) infection. Our previous work on Nox4 induction by transforming growth factor-beta (TGF-beta) highlighted critical roles of this oxidase in fibrotic and metastatic disease, suggesting Nox4 inhibitors have important therapeutic potential: 1) We showed hepatitis C virus (HCV) infection of hepatocytes occurs through an autocrine TGF-beta pathway, which suggested links between excess Nox4-derived ROS production during chronic HCV infection and hallmarks of liver disease progression (fibrosis, cirrhosis, and hepatocellular carcinogenesis). 2) We showed Nox4 induction by TGF-beta in many cell types is a critical event in the epithelial-to-mesenchymal transition (EMT), leading to increased cell plasticity at the onset of profibrotic programs involving Nox4-dependent fibronectin synthesis and cell motility. 3) We described divergent effects of wild type versus mutant p53 on TGF-beta/SMAD3-mediated Nox4 induction during EMT and cancer cell invasion and metastasis. We showed WT-p53 is a potent suppressor of TGF-beta-induced Nox4, ROS production, and cell migration, whereas two metastatic tumor-associated mutant p53 proteins (R175H or R280K) caused enhanced Nox4 expression and cell migration (Boudreau (2014) Br J Cancer 110:2569-82). We have now expanded these observations by surveying functional effects of common p53 mutant forms detected in many cancers (R248Q, R249S, R273H, and D281G). We tested the effects of these mutant proteins in three types of epithelial cancers (breast, lung and hepatocytes) and confirmed the importance of this TGF-beta-SMAD3-mutantp53-Nox4 axis in cell migration and tumor metastasis. Ongoing studies are correlating p53 mutation status with Nox4 in primaries tumors. Since the discovery of the convergence of TGF-beta and mutant p53 pathways promoting pro-migratory and pro-metastatic phenotypes, identification of downstream targets with therapeutic potential is of high interest. Thus, other efforts are aimed at defining transcriptional mechanisms involved in mutant p53 and TGF-beta/SMAD3-regulated Nox4 gene expression as well as subsequent Nox4-dependent redox signaling mechanisms involved in cell migration. Given the prevalence of p53 mutations with cancer progression (>50% of all cancers), we have initiated studies with collaborators in the National Center for Advancing Translational Sciences with aims of identifying Nox4 inhibitors as potential anti-metastatic agents. Here we are developing inducible Nox4 expressing cell lines for high-throughput oxidase inhibition assays along with assays of inhibitors of Nox4-dependent migration with several metastatic tumor models. Other Nox4-related studies were initiated with collaborators in the National Institute on Minority Health and Health Disparities focusing on the potential importance of Nox4 induction in type-2 diabetes within minority (African American) populations. Here we showed TGF-beta and insulin signaling pathways converge to synergistically enhance Nox4 expression and ROS production and will explore whether these changes promote profibrotic and proiflammatory disease processes. Previous studies have already defined genetic polymorphisms linked to enhanced TGF-beta activation pathways in African Americans, thus targeting this pathway may be particularly relevant in diabetic disease complications, including increased risks for obesity, cancer, and kidney disease in this affected population. Nox1 is another oxidase involved in cell migration, wound healing, and colon epithelial homeostasis, in this case induced by inflammatory cytokines (tumor necrosis factor (TNF)-alpha or interferon-gamma) and activated by microbial formyl peptides. Our current studies identified Prdx6 as a novel positive regulator of ROS generation by Nox1 in several cell models and showed Prdx6 supports Nox1-dependent migration of the colon epithelial tumor line, HCT-116. Initially, we demonstrated Prdx6 binds to Noxa1 in yeast two-hybrid screening experiments aimed at identifying novel binding partners of the Noxa1 SH3 domain. Then we showed Prdx6 associates with the assembled Nox1 complex in several cell models. Experiments manipulating cellular Prdx6 levels confirmed its close association with at least two other Nox1 components (Noxa1 and Noxo1) by showing RNAi suppression of Prdx6 production reduced Noxa1 and Noxo1 levels and Nox1 activity, whereas overexpression of Prdx6 led to higher levels of these Nox1 components and higher ROS generation. Furthermore, we showed induction by TNF-alpha stabilizes higher levels of Prdx6 along with Noxa1 and Noxo1. Finally, we showed the Nox1-supportive effects of Prdx6 require both its glutathione peroxidase and PLA2 activities to support higher Nox1 activity or Nox1-dependent cell migration. These findings provide further insight on post-translation regulation of this oxidase that may be relevant to proposed physiological and pathological roles of Nox1 in vascular, epithelial, and tumor tissues. In the third research area we examined airway epithelial cell responses to influenza A virus (IAV) infection by PR8/34(H1N1) and HK8/68 (H2N3) strains. We showed Duox1 is the prevalent NADPH oxidase detected in murine and human tracheal epithelial cells and in primary airway cells differentiated in air-liquid interface (ALI) cultures. We observed decreased Duox1 expression and lower hydrogen peroxide release in ALI cultures 24-48 hours infection after with either PR8/34 or HK8/68 IAV. Our earlier studies showed Duox-derived hydrogen peroxide production in ALI cultures supports the microbicidal activity of lactoperoxidase against several bacterial pathogens. Therefore, Duox1 suppression following IAV infection may predispose airways to subsequent bacterial infection by compromising Duox- and lactoperoxidase-dependent microbial killing. Future work will test this hypothesis using the Duox1-deficient mouse. Other basic studies identified structural epitopes critical in the maturation and transport of the Duox-Duox activator complex to the plasma membrane, where it functions as a dedicated hydrogen peroxide generator on cell surfaces.